In Maxwell's famous Gedanken (thought) experiment, a demon operates a door between two boxes of gas at the same temperature. The demon sorts the molecules keeping the faster molecules in one box and the slower in the other, violating the basic laws of thermodynamics. This paradox has since been resolved in many different ways. Leff, H. S. and Rex, A. F. (1990), "Resource letter md-1: Maxwell's demon," Am. J. Physics 58:201-209.
A similar arrangement can be used to separate particles. Consider a mixture of particles of two different sizes suspended in water in one box and pure water in the other. If the demon opens and closes the door between the boxes quickly enough so that none of the larger particles have time to diffuse through the doorway, but long enough so that some of the smaller particles have enough time to diffuse into the other box, some separation will be achieved.
Recently two experiments have been done where a spatially asymmetric potential is periodically applied in the presence of a number of Brownian particles. Faucheux, L. S. et al. (1995), "Optical thermal ratchet," Physical Rev. Letters 74:1504-1507; Rousselet, J. et al. (1994), "Directional motion of Brownian particles induced by a periodic asymmetric potential," Nature 370:446-448.
This has been shown to lead to a directed motion of the particles at a rate depending on the diffusion coefficient. One experiment (Rousselet, J. et al. (1994), "Directional motion of Brownian particles induced by a periodic asymmetric potential," Nature 370:446-448) used microfabricated electrodes on a microscope slide to apply an electric field for the potential. This idea is also the subject of European Patent Publication 645169 of Mar. 29, 1995, for "Separation of particles in a fluid--using a saw-tooth electrode and an intermittent excitation field," Adjari, A. et al. The other experiment (Faucheux, L. S. et al. (1995), "Optical thermal ratchet," Physical Rev. Letters 74:1504-1507) used a modulated optical tweezer arrangement.
Diffusion is a process which can easily be neglected at large scales, but rapidly becomes important at the microscale. The average time for a molecule to diffuse across a distance d is 2t=d.sup.2 /D where D is the diffusion coefficient of the molecule. For a protein or other large molecule, diffusion is relatively slow at the macro-scale (e.g., hemoglobin with D equal to 7.times.10.sup.-7 cm.sup.2 /s in water at room temperature takes about 10.sup.6 seconds (ten days) to diffuse across a one centimeter pipe, but about one second to diffuse across a ten micron channel).
Using tools developed by the semiconductor industry to miniaturize electronics, it is possible to fabricate intricate fluid systems with channel sizes as small as a micron. These devices can be mass-produced inexpensively and are expected to soon be in widespread use for simple analytical tests.
A process called "field-flow fractionation" (FFF) has been used to separate and analyze components of a single input stream in a system not made on the microscale, but having channels small enough to produce laminar flow. Various fields, including concentration gradients, are used to produce a force perpendicular to the direction of flow to cause separation of particles in the input stream. See, e.g., Giddings, J. C., U.S. Pat. No. 3,449,938, Jun. 17, 1969, "Method for Separating and Detecting Fluid Materials;" Giddings, J. C., U.S. Pat. No. 4,147,621, Apr. 3, 1979, "Method and Apparatus for Flow Field-Flow Fractionation;" Giddings, J. C., U.S. Pat. No. 4,214,98 1, Jul. 29, 1980), "Steric Field-Flow Fractionation;" Giddings, J. C., et al., U.S. Pat. No. 4,250,026, Feb. 10, 1981, "Continuous Steric FFF Device for The Size Separation of Particles;" Giddings, J. C., et al., (1983), "Outlet Stream Splitting for Sample Concentration in Field-Flow Fractionation," Separation Science and Technology 18:293-306; Giddings, J. C. (1985), "Optimized Field-Flow Fractionation System Based on Dual Stream Splitters," Anal. Chem. 57:945-947; Giddings, J. C., U.S. Pat. No. 4,830,756, May 16, 1989, "High Speed Separation of Ultra-High Molecular Weight Polymers by Hyperlayer Field-Flow Fractionation;" Giddings, J. C., U.S. Pat. No. 4,141,651, Aug. 25, 1992, "Pinched Channel Inlet System for Reduced Relaxation Effects and Stopless Flow Injection in Field-Flow Fractionation;" Giddings, J. C., U.S. Pat. No. 5,156,039 Oct. 20, 1992, "Procedure for Determining the Size and Size Distribution of Particles Using Sedimentation Field-Flow Fractionation;" Giddings, J. C., U.S. Pat. No. 5,193,688, Mar. 16, 1993, "Method and Apparatus for Hydrodynamic Relaxation and Sample Concentration in Field-Flow Fraction Using Permeable Wall Elements;" Caldwell, K. D. et al., U.S. Pat. No. 5,240,618, Aug. 31, 1993, "Electrical Field-Flow Fractionation Using Redox Couple Added to Carrier Fluid;" Giddings, J. C. (1993), "Field-Flow Fractionation: Analysis of Macromolecular, Colloidal and Particulate Materials," Science 260:1456-1465; Wada, Y., et al., U.S. Pat. No. 5,465,849, Nov. 14, 1995, "Column and Method for Separating Particles in Accordance with their Magnetic Susceptibility." None of these references disclose the use of a separate input stream to receive particles diffused from a particle-containing input stream.
A related method for particle fractionation is the "Split Flow Thin Cell" (SPLITT) process. See, e.g., Williams, P. S., et al. (1992), "Continuous SPLITT Fractionation Based on a Diffusion Mechanism," Ind. Eng. Chem. Res. 31:2172-2181; and J. C. Giddings U.S. Pat. No. 5,039,426. These publications disclose devices with channels small enough to produce laminar flow, but again only provide for one inlet stream. A further U.S. patent to J. C. Giddings, U.S. Pat. No. 4,737,268, discloses a SPLITT flow cell having two inlet streams; however the second inlet stream is not an indicator stream, but rather a particle-free stream. Giddings U.S. Pat. No. 4,894,146 also discloses a SPLITT flow cell having two input streams, but no indicator stream. All these SPLITT flow methods require the presence of more than one output stream for separating various particle fractions.
None of the foregoing publications describes a channel system device capable of analyzing small particles in very small quantities of sample which may also contain larger particles, particularly larger particles capable of affecting the indicator used for the analysis. No devices or methods using indicator streams within the cell system device are described.
Microfluidic devices allow one to take advantage of diffusion as a rapid separation mechanism, which also allows for efficient and precise detection of the separated (diffused) particles. Flow behavior in microstructures differs significantly from that in the macroscopic world. Due to extremely small inertial forces in such structures, practically all flow in microstructures is laminar. This allows the movement of different layers of fluid and particles next to each other in a channel without any mixing other than diffusion. On the other hand, due to the small lateral distances in such channels, diffusion is a powerful tool to separate molecules and small particles according to their diffusion coefficients, which is generally a function of their size. A sample stream can be in laminar flow with a stream containing an indicator substance, which provides a means for detecting an analyte which has diffused from the sample stream into the indicator stream.
Weigl, B. H. and Yager, P. "Silicon-Microfabricated Diffusion-Based Optical Chemical Sensor," Sensors & Actuators B--"Europetrode" (Conference) Apr. 2, 1996, Zurich, Switzerland; Weigl, B. H., Holl, M. A., Schutte, D., Brody, J. P., and Yager, P. "Diffusion-Based Optical Chemical Detection in Silicon Flow Structures," Analytical Methods and Instrumentation, .mu.TAS 96 special edition, 1996; Weigl, B. H., van den Engh, G., Kaiser, R., Altendorf, E., and Yager, P. "Rapid Sequential Chemical Analysis Using Multiple Fluroescent Reporter Beads," .mu.TAS 96, Conference Proceedings, 1996; Weigl, B. H., Hixon, G. T., Kenny, M., Zebert, D., Dwinnell, S., Buj, T. and Yager, P. "Fluorescence Analyte Sensing in Whole Blood Based on Diffusion Separation in Silicon-Microfabricated Flow Structures, SPIE Proceedings, Feb. 9-11, 1997, J. Lakowitz (ed.), Fluorescence Sensing Technology III; and Brody, J. and Yager, P. "Low Reynolds Number Micro-Fluidic Devices," Solid State Sensor and Actuator Workshop, Hilton Head, S.C. Jun. 2-6, 1996, all of which are incorporated by reference in their entirety, describe various devices and methods utilizing laminar flow and diffusion principles to detect the presence of and determine the concentration of various analytes in samples, e.g., whole blood.
U.S. patent application Ser. No. 08/625,808 "Microfabricated Diffusion-Based Chemical Sensor," filed Mar. 29, 1996 (now U.S. Pat. No. 5,716,852, issued on Feb. 10, 1998), U.S. patent application Ser. No. 08/829,679 (Attorney Docket No. 6-96A) "Microfabricated Diffusion-Based Chemical Sensor," filed Mar. 31, 1997 now U.S. Pat. No. 5,972,710 issued on Oct. 26, 1999 and P.C.T. Patent Application Serial No. PCT/US 97/05245 "Microfabricated Diffusion-Based Chemical Sensor," filed Mar. 31, 1997, each of which is hereby incorporated in its entirety by reference herein, disclose a microfabricated device comprising a laminar flow channel, at least two inlets in fluid connection with the laminar flow channel for conducting into the flow channel an indicator stream and a sample stream, and an outlet. Smaller particles in the sample stream diffuse into the indicator stream, forming a detection area wherein measurements of a detectable property are made. These three applications disclose methods for determining the concentration of analytes in a sample stream by detecting the position within the laminar flow channel of analyte particles from the sample stream diffusing into the indicator stream causing a detectable change in the indicator stream. Alternatively, the position within the laminar flow channel of the region where equilibrium of diffusion of the analyte from the sample stream into the indicator stream has occurred can be used to determine analyte concentration. Additionally, information useful for determining the concentration of analyte particles in the sample stream may be obtained by providing means such as specimen channels for conducting specimen streams from the indicator stream at successive intervals along the length of the laminar flow channel. Changes in the intensity of the signals from specimen channel to specimen channel may be used to calculate the concentration of analyte particles in the original sample.
The devices of these three applications can be in fluid connection with a device such as that disclosed in U.S. patent application Ser. No. 08/534,515 "Silicon Microchannel Optical Flow Cytometer," filed Sep. 27, 1995, now U.S. Pat. No. 5,726,751, issued on Mar. 10, 1998, which is incorporated in its entirety by reference herein, which allows for single-file flow of particles and which provides reflective surfaces for detection of reflected light rather than transmitted light. The devices of these three applications can alternatively be in fluid connection with a sheath flow module as disclosed in U.S. patent application Ser. No. 08/823,747 "Device and Method for 3-Dimensional Alignment of Particles in Microfabricated Flow Channels," filed Mar. 26, 1997, which is hereby incorporated in its entirety by reference herein.
The use of quality control materials and internal standard materials is, of course, well known in the art. See L. A. Kaplan, Clinical Chemistry, 1989, 2nd ed., The C. V. Mosby Co., St. Louis, which is incorporated by reference herein to the extent that it is not inconsistent with the disclosure herein, for a discussion of previously known methods and materials for quality control, internal standards, and calibrators. The purpose of quality control of analytical testing is to make certain that each measurement performed on a sample is reliable. In general, in previously known methods of using quality control materials, as described in Kaplan's Clinical Chemistry, p. 278, a control is measured daily (or once per shift) and can be plotted in a Levy-Jennings plot which graphs the established target average.+-.two standard deviations (on the y-axis) versus time, e.g., the days of the month (on the x-axis). The target value is the estimated concentration of the analyte of interest in the sample within a certain degree of accuracy, because on any given day, the measured control value will differ slightly within the specifications of the instrument, calibration, etc. The user must establish a target value for each analyte by regular laboratory procedures known in the art. However, the measured control value should be fairly constant over time. If the control values slowly drift down or up for the target value, then this indicates a trend. If the control values show a sudden jump in the values recorded from one average to another, then this indicates a shift. If a systematic bias is indicated (a change in accuracy) or a change in precision is indicated, the user must check the reagents, internal standards, and instrumentation.
As described in Kaplan's Clinical Chemistry, the purpose of internal standards is to improve accuracy and precision as well as providing a check of quality control because for a given internal standard batch, its measured value (e.g., detectable property correlatable to concentration, as determined by any of various detecting means) should be the same over time.
In previously known methods and materials for quality control and using internal standards, quality control materials were different from those used for internal standards.